1. Does induction of the competence regulon increase survival even when no DNA is available?
To explain why the CRP-S competence regulon includes genes that are unlikely to play any role in DNA uptake, we've hypothesized that these genes are turned on because they help the cell cope with stalled replication forks until the nucleotide supply can be restored. If that's right, then a reasonable prediction is that viability should be reduced or growth delayed by that conditions that strongly induce the CRP-S regulon should threaten cell viability or growth, and that preventing CRP-S induction under these conditions will reduce viability or growth.
That is, mutants that can't turn on the CRP-S regulon should lose viability or show delayed growth when they experience competence-inducing conditions, but cells that can turn the genes on should do better. So I'm going to grow up our sxy-knockout mutant and carefully compare it to sxy+ cells for survival in MIV, the starvation medium that best induces competence. As a control, I'll compare its growth in the two defined RM media (+ and - inosine).
We now have a new purR knockout that we're sure is a complete knockout (because almost the whole gene has been deleted). I need to check its competence, so I'll also check how well it survives transfer to MIV. Maybe I'll also make a purR sxy double knockout and check that.
2. Does the ability to synthesize purine nucleotides affect competence development?
We now have a purH knockout mutant, which can't synthesize purine nucleotides and can't grow in RM medium unless inosine is provided). Does it develop normal competence when transferred to MIV from RM+ or from rich medium? How quickly does it die in MIV? What if it can't turn on the competence regulon - does it die sooner?
3. Does the citrulline in MIV make any contribution as a pyrimidine precursor?
From reading the very old papers about H. influenzae's nutrient requirements, I've found out that it can use citrulline to synthesize the amino acid arginine as well as pyrimidine nucleotides. That is, cells can't synthesize either arginine or pyrimidines from scratch. If given uracil as a pyrimidine precursor , they must also be given arginine. But if they're given citrulline they don't need the arginine. I also hadn't realized that MIV contains citrulline, which cells could use to synthesize pyrimidine nucleotides. But the concentration is only 6 µg/ml, whereas MMB and the RPMI-based medium both contain 300 µg/ml.